AD Award Number: Molecular Basis of Autophagic Cell Death in … · 2018. 11. 8. · I, Ramesh...

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W81XWH-08-1-0183

TITLE:

Molecular Basis of Autophagic Cell Death in Prostate Cancer

PRINCIPAL INVESTIGATOR:

Ramesh Kaini

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University of New Mexico

Albuquerque, NM, 87131-0001

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W81XWH-08-1-0183

Molecular Basis of Autophagic Cell Death in Prostate Cancer

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6. AUTHOR(S) Ramesh Kaini

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Albuquerque, NM, 876131-0001

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14. ABSTRACT To understand the molecular basis of autophagy in prostate cancer, I am trying to isolate

pure autophagosomes from autophagic cell survival and autophagic cell death Prostate cancer

cell model and profile proteins and lipids to identify autophagy abundant/specific proteins

and lipid molecule that are essential for functional autophagy in prostate cancer. I

established EGFP.LC3 stably expressing LNCaP cell line and characterized the autophagic

activity by western blot, translocation assay and Atg5 siRNA transfection. I also confirmed

the autophagic activity in PC3.EGFP.LC3 cells. I was able to enrich autophagosomes using

gradient ultracentrifugation and currently trying to optimize the condition for further

purification by Immunopurification using beads.

15. SUBJECT TERMS Autophagy, Prostate cancer, Subcellular Organelle Purification

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Table of Contents

Page

Introduction…………………………………………………………….………..….. 4

Body………………………………………………………………………………….. 4

Key Research Accomplishments………………………………………….…….. 8

Reportable Outcomes……………………………………………………………… 8

Conclusion…………………………………………………………………………… 8

References……………………………………………………………………………. 8

Appendices…………………………………………………………………………… 9

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Introduction: I, Ramesh Kaini, PI of the project ( Title: Molecular Basis of Autophagic Cell Death, contract no.: W81XWH-08-1-0183); was awarded FY07 Prostate Cancer Training Award ( Pre-doctoral -PhD ) to pursue my career in prostate cancer research. The purpose of the award is to support me to understand the molecular basis of autophagy and its role in cell survival and cell death of prostate cancer cells in response to different stimuli. My focus is to isolate autophagosomes from both autophagic cell death and autophagic cell survival prostate cancer cell models and profile proteomes and lipidomes to identify novel autophagy abundant and/or specific protein and lipid molecules that are essential for functional autophagy in prostate cancer cells. The project consists of three aims : (i) to create autophagic cell death and autophagic cell survival models in LNCaP (P53+/+) and PC-3 (P53-/-) cells; (ii) to purify autophagy abundant and specific vacuoles, autophagosomes and autolysosome; and (iii) to profile and characterize membrane protein and lipid species of autophagosomes and autolysosome using quantitative proteomic and lipidomic approaches. Body: A. Training Accomplishments: This year, I mainly focused to update my knowledge and expertise in cancer biology, autophagy and required techniques. Notable was my “The Edward A. Smuckler Memorial Workshop” award to attend the “AACR Workshop in Cancer Research: Pathobiology of Cancer” at Snowmass village, CO from July 6-13, 2008. The intense one week long workshop was very helpful for me to widen my knowledge in cancer pathogenesis at molecular and morphologic level from some of the best scientists working in the field. I also continued to actively participate in CMBD seminar, departmental journal club, pathology grand round and cancer center visiting professor lectureship. These seminars/ journal club / grand rounds continue to help me to widen my knowledge in multiple disciplines of biomedical science. Besides, participation in regular prostate cancer interest group meeting was also productive for me to update with recent advances in prostate cancer research. Throughout year, my mentor Dr. Andy Hu was very supportive in providing guidance and research support. He was always there when I need help. He was also very keen on helping me to develop professionalism. This year, he also provided me an opportunity to assist in peer-reviewing an article. It was a great experience for me to critically analyze a review paper and provide comments in a professional way. My co-mentor, Dr. Laurel Sillerud was also very supportive to guide me through the problems encountered during research. I have also regular interaction with other advisors, Dr. Bisoffi & Dr. Deretic. This year, I learned and applied the techniques, gradient ultracentrifugation and confocal microscopy. I also learned the principle and sample preparation for electron microscopy and flow cytometry. As of Fall 2008; I have earned 88 credit hours with cumulative GPA of 3.87.

B. Research Accomplishments:

There are three specific aims of my project as outlined previously in the introduction. The target was to achieve Specific Aim 1 and 2 in the first two years and Specific Aim 3 in the third year.

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Accomplishments for Aim 1: I successfully transfected the LNCaP cells with pEGFP.LC3 plasmid construct (gift from Dr. Noburu Mizushima) and generated stably transfected clone of LNCaP cells. Stably transfected cells were selected by continuously growing the transfected cells under G418 selective pressure ( 400 ug/ml). During autophagy, cytosolic EGFP.LC3 are processed and targeted to newly forming autophagosomes which are observed as distinct puncta under the fluorescence microscopy (1-2). I induced autophagy by incubating LNCaP.EGFP.LC3 cells in starvation medium and assessed autophagy by observing the translocation of diffused cytosolic recombinant EGFP.LC3 protein to distinct puncta ( Fig. 1A,B ). Autophagic activity was quantified by calculating the percentage of cells showing distinct puncta over different time points of starvation ( Fig 1C). Similar results were observed in pEGFP.LC3 stably transfected PC3 prostate cancer cell line ( obtained from Dr. wei-Pang Hwang).

Figure 1. Starvation induces autophagy formation in LNCaP- EGFP.LC3 cells. (A) Cytosolic localization of EGFP.LC3 in stably transfected LNCaP cells. (B) Translocation of EGFP.LC3 in puncta after incubation in HBSS in LNCaP-EGFP.LC3 cells. (C) Quantitation of EGFP.LC3 puncta formation in stably transfected LNCaP cells at different time points after incubation in HBSS. Cells were grown in cover slip and then incubated in HBSS as noted. Six visual fields were selected in random for each group. All cells in each field were counted (minimum number of 350 cells) and the percentage of cells with puncta formation was calculated.

Exogenously expressed EGFP.LC3 can sometime aggregate and show distinct puncta formation independent of autophagic activity (2,3). Atg5 is a key molecule required for the formation of autophagosomes(1-3). I did Atg5 siRNA transfection in EGFP.LC3 stably expressing LNCaP cells and observed that knocking down of Atg5 abrogated the translocation of cytosolic EGFP.LC3 to distinct puncta ( fig 2) . This confirmed that my EGFP.LC3 transfected cells are bonafide autophagic model.

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Figure 2. ATG5 knockdown abrogates autophagy formation in LNCaP-

EGFP.LC3 cells. Transfection of ATG5 siRNA was performed using the Lipofectamine 2000 and the supplied protocol. Translocation assay was performed 48 hours post-transfection. ATG5 siRNA at 25 nM concentration reduces the autophagy formation by starvation in HBSS.

Currently, I am working forward to characterize proposed autophagic cell survival model by using sulforaphane. ApoL1 induced autophagic cell death is defined in the attached two co-authored papers (Appendices 1, 2).

Accomplishments for Aim 2: To establish the method for isolation and purification of autophagosomes from LNcaP.EGFP.LC3 cells and PC3.EGFP.LC3 cells induced by starvation in HBSS medium (pro-survival); I initially enriched the autophagic fraction by differential centrifugation. I biotinylated the antibodies against LC3 and conjugated with streptavidin coated magnetic beads. Then, the enriched autophagic fraction was incubated with Anti-LC3-Mag-Nap and the bound organelle fraction was washed three times. However, thus purified autophagosomes was not pure enough for downstream proteomic and lipidomic study on multiple repeated attempts. As discussed earlier, the recombinant protein EGFP.LC3 translocates to autophagic membrane during autophagosomes formation. So, I used antibody against EGFP to selectively immunopurify autophagosomes, but the result was similar to that obtained by using antibody against LC3. Intracellular lysosomes have been purified using flow cytometry assisted organelle sorting (FAOS) (4). To take advantage of selective localization of EGFP.LC3 in the autophagosomes; I tried to sort out autophagosomes using flow cytometry assisted organelle sorting as an alternative method to

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isolate autophagosomes. However, the signal was not enough to detect by the flow cytometry and that approach was aborted.

Another promising method to purify intracellular organelles is gradient ultracentrifugation. I tried to enrich autophagosomes by using sucrose gradient followed by immunopurification using antibodies tagged with magnetic particles. Previously, rat liver autophagosomes were also purified by using nycodenz/ percoll gradient (6). I attempted to imply the same methods to purify autophagosomes. Both methods work to highly enrich the autophagosomes.

Figure 3: Electron-micrograph of isolated autophagosomes using Anti-EGFP-Mag-Nap after separation of intracellular oraganelles using sucrose gradient ultracentrifugation. Beads , autophagosomes and contaminating organelles are seen in the picture.

I’m further trying to optimize the condition for Immunopurification of these purified autophagosomes from density gradient centrifugation, by using antibody against LC3 or EGFP that are conjugated with beads. I hope, immunopurification after the successful density gradient ultracentrifugation will yield enough pure autophagic samples for my downstream proteomic and lipidomic study. Based on contamination level, I plan to imply negative selection of contaminants before positive selection of autophagosomes using beads tagged with antibody against the molecular marker of contaminating organelles.

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Key Research Accomplishments: # Successfully established stably transfected LNCaP.EGFP.LC3 cells. # Characterized and confirmed the autophagic activity in LNCaP.EGFP.LC3 cells by western blot, translocation assay and Atg5 siRNA transfection experiment. # Confirmed autophagic activity in PC3.EGFP.LC3 cells. # Co-authored and documented ApoL1 induced autophagic cell death model. # Obtained purified autophagosomes from gradient ultracentrifugation. Reportable Outcomes: 1. LNCaP.EGFP.LC3 cells. 2. Wan G, Zhaorigetu S, Liu Z, Kaini R, Jiang Z, Hu CA: Apolipoprotein L1, a novel Bcl-2 homology domain 3-only lipid-binding protein, induces autophagic cell death. J Biol Chem 2008; 283(31):21540-9. 3. Zhaorigetu S, Wan G, Kaini R, Jiang Z, Hu CA: ApoL1, a BH3-only lipid-binding protein, induces autophagic cell death. Autophagy 2008; 4(8):1079-82. Conclusion: I completed the most part of Aim 1 and am currently trying to optimize the purification of autophagosomes using density gradient and immunopurification. I’m hopeful that I’ll complete the remaining part on Aim 1 and Aim 2 before the completion of year 2 of funding as targeted. I also widened my breadth of knowledge a lot on prostate cancer, cancer pathogenesis and other discipline of biomedical science through various workshops, didactic journal club/ seminars/ grand rounds and group meeting. References: 1. Mizushima N, Ohsumi Y, Yoshimori T: Autophagosome formation in mammalian cells. Cell Structure & Function 2002; 27:421-429 2. Klionsky DJ et al : Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4(2): 151-175

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http://www.ncbi.nlm.nih.gov/pubmed/18505729?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/18505729?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/18927493?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/18927493?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

3. Ciechomska IA & Tolkovsky AM: Non-autophagic GFP-LC3 puncta induced by saponin and other detergents. Autophagy 2007; 3(6) : 586-590 4. Rajotte D, Stearns CD, Kabcenell AK : Isolation of mast cell secretory lysosomes using flow cytometry. Cytometry 2003; 55A(2): 94-101

5. Strømhaug PE, Berg TO, Fengsrud M, Seglen PO : Purification and characterization of autophagosomes from rat hepatocytes. Biochem J 1998; 335(2): 217-224

Appendices

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http://www.ncbi.nlm.nih.gov.libproxy.unm.edu/pubmed/9761717?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov.libproxy.unm.edu/pubmed/9761717?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov.libproxy.unm.edu/pubmed/9761717?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

Apolipoprotein L1, a Novel Bcl-2 Homology Domain 3-onlyLipid-binding Protein, Induces Autophagic Cell Death*Received for publication, January 9, 2008, and in revised form, May 26, 2008 Published, JBC Papers in Press, May 26, 2008, DOI 10.1074/jbc.M800214200

Guanghua Wan, Siqin Zhaorigetu, Zhihe Liu, Ramesh Kaini, Zeyu Jiang, and Chien-an A. Hu1

From the Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine,Albuquerque, New Mexico 87131

The Bcl-2 family proteins are important regulators of type Iprogrammed cell death apoptosis; however, their role in auto-phagic cell death (AuCD) or type II programmed cell death isstill largely unknown.Herewe report the cloning and character-ization of a novel Bcl-2 homology domain 3 (BH3)-only protein,apolipoprotein L1 (apoL1), that, when overexpressed and accu-mulated intracellularly, induces AuCD in cells as characterizedby the increasing formation of autophagic vacuoles and activat-ing the translocation of LC3-II from the cytosol to the autoph-agic vacuoles. Wortmannin and 3-methyladenine, inhibitors ofclass III phosphatidylinostol 3-kinase and, subsequently, auto-phagy, blocked apoL1-induced AuCD. In addition, apoL1 failedto induceAuCD in autophagy-deficientATG5�/� andATG7�/�mouse embryonic fibroblast cells, suggesting that apoL1-in-duced cell death is indeed autophagy-dependent. Furthermore,a BH3 domain deletion construct of apoL1 failed to induceAuCD, demonstrating that apoL1 is a bona fide BH3-only pro-death protein. Moreover, we showed that apoL1 is inducible byp53 in p53-induced cell death and is a lipid-binding proteinwithhigh affinity for phosphatidic acid (PA) and cardiolipin (CL).Previously, it has been shown that PA directly interacted withmammalian target of rapamycin and positively regulated theability of mammalian target of rapamycin to activate down-stream effectors. In addition, CL has been shown to activatemitochondria-mediated apoptosis. Sequestering of PA and CLwith apoL1 may alter the homeostasis between survival anddeath leading to AuCD. To our knowledge, this is the first BH3-only proteinwith lipid binding activity that, whenoverproducedintracellularly, induces AuCD.

Bcl-2 family members play crucial roles in regulating pro-grammed cell death (PCD).2 In stressed cells the live-or-die

decision is largely determined by interplay between opposingmembers of the Bcl-2 protein family (1–4). Eachmember of theBcl-2 family contains at least one of four conserved amino acid(aa) sequences, Bcl-2 homology (BH) domains 1–4. Based ontheir function in PCD, the Bcl-2 familymembers can be dividedinto three subgroups as follows: multi-BH domain anti-death,multi-BH domain pro-death, and BH3-only pro-death (1–3).The BH3 domain is conserved across all Bcl-2 family membersand is the only domain retained by BH3-only proteins, furtheremphasizing the functional significance of this domain (4). Inmammals, there are two major types of PCD, apoptosis andautophagic cell death (AuCD). Apoptosis (type 1) is character-ized by nuclear condensation/DNA fragmentation and cellmembrane alteration, without major ultrastructural changes ofsubcellular organelles. It usually involves the activation ofcaspases as well as the release of apoptogenic effectors, such ascytochrome c and apoptosis-inducing factor, from mitochon-dria that contribute to the acquisition of the apoptotic mor-phology (5–8). Themolecular basis of AuCD (type 2), however,is not as well characterized in mammalian cells, even thoughmany lines of evidence connect autophagy to human disease.Autophagy is generally defined as a lysosome-dependentmech-anism of intracellular degradation that is used for the turnoverof cytoplasmic constituents. There are three major classifica-tions of autophagy: macroautophagy, microautophagy, andchaperone-mediated autophagy (9–12). Macroautophagy(hereafter referred to as autophagy) is a highly conservedeukaryotic catabolic process involving the sequestration of thecargo (i.e. fraction of cytosol or organelles) within doublemem-branes, creating autophagosomes, which then fuse with theendosome and lysosome to create autolysosomes. In the lumenof autolysosomes, lysosomal enzymes operate at low pH tocatabolize the autophagic material (12–14). Autophagosomesand autolysosomes, also called autophagic vacuoles (AV), areconsidered to be de novo subcellular organelles specific tothe autophagic process. Autophagy allows cells to eliminatelarge portions of the cytoplasm and is directly involved toeliminate aberrant protein aggregates, remove damaged/

* This work was supported, in whole or in part, by National Institutes of HealthGrant 5RO1CA106644-01A1 from NCI and New Mexico-IdeA Network ofBiomedical Research Excellence (NM-INBRE) Program P20RR016480-04 (toC.-A. A. H.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 To whom correspondence should be addressed: Dept. of Biochemistryand Molecular Biology, MSC08 4670, 1 University of New Mexico, Albu-querque, NM 87131-0001. Tel.: 505-272-8816; Fax: 505-272-6587;E-mail: [email protected].

2 The abbreviations used are: PCD, programmed cell death; AuCD, autoph-agic cell death; AV, autophagic vacuoles; BH, Bcl-2 homology domain; CL,cardiolipin; LC3, microtubule associated proteins light chain 3; PA, phos-phatidic acid; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-phos-phate kinase; PIP, phosphoinositide; 3-MA, 3-methyladenine; PARP, poly-

(ADP-ribosyl) polymerase; TEM, transmission electron microscopy; aa,amino acid; mTOR, mammalian target of rapamycin; MEF, mouse embry-onic fibroblast; RT, reverse transcription; BSA, bovine serum albumin;HBSS, Hanks’ balanced salt solution; PE, phosphoethanolamine; PG, phos-phoglycerol; PI, phosphatidylinositol; SM, sphingomyelin; TG, triglyceride;AD, adenovirus; Dox, doxycycline; HDL, high density lipoprotein; PI-3,4,5-P3, phosphatidylinositol 3,4-trisphosphate; PI-3,4-P2, phosphatidylinositol3,4-bisphosphate; PI3P, phosphatidylinositol 3-phosphate; PI4P, phospha-tidylinositol 4-phosphate; Ptd, phosphatidylinositol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 31, pp. 21540 –21549, August 1, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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aged organelles, defend against pathogen invasions, and coveran essential role in early neonatal development and differenti-ation (15–16). Upwards of 20 autophagy-related genes (ATG)have been identified, which regulate and/or directly participatein the autophagic pathway, particularly in AV formation (10,13, 14). Deficiency of ATGs for example, ATG5 and ATG7,causes loss of AV formation and subsequently lack of autoph-agic response. These deficiencies correlate with various dis-eases such as cancer, cardiomyopathy, and neurodegenerativediseases (17–22). Paradoxically, recent evidence suggests thatautophagy is, on the one hand, a survival pathway necessary tosustain mammalian viability during periods of starvation, andon the other hand, progressive autophagy can be ameans to celldeath (7, 11, 12). Significantly, Berry and Baehrecke (23)reported that autophagy is involved in physiological cell deathduring Drosophila development but is controlled by similarmechanisms as those that control autophagy function in cellsurvival. Furthermore, they provide in vivo evidence that bothgrowth arrest and autophagy are required for physiologicalAuCD. However, the molecular determinants that dictate thecontrol of autophagy for cell survival or death have not yet beenrevealed.Aside from their role in apoptosis, the members of the Bcl-2

family also play an important role in autophagy. For example,when treating apoptosis-deficient cells, such as BAX and BAKdouble knock-out cells, with cytotoxic compounds (for exam-ple, etoposide), the primary response of those cells was AuCD,suggesting that homeostatic states of these knock-out Bcl-2members play pivotal roles in regulating AuCD (24–27). Inaddition, it has been shown that the anti-death Bcl-2 andBcl-xLcan bind and inhibit Beclin1, a mammalian ortholog of yeastATG6, and subsequently inhibit autophagy (28, 29). Overex-pression of Beclin1 has been shown to induce formation of AVand autophagy in mammalian cells (28). Interestingly, Beclin1has been found to be a haploinsufficient tumor suppressor fre-quently mutated/deleted in breast, prostate, and ovarian can-cers (12, 30, 31). Heterozygous knock-out mice of Beclin1(Beclin1�/�) are tumor-prone, suggesting that autophagy has atumor suppression function (30). Importantly, structural andfunctional analyses showed that Beclin1 is a novel BH3-onlyprotein, and this BH3 domain within Beclin1 mediates associ-ations with Bcl-2 and Bcl-xL (32–34). In addition to Beclin1,recent studies showed other BH3-only members that also playregulatory roles in autophagy in physiological context. Forexample, Bid, a BH3-only protein, showed involvement in reg-ulating both apoptosis and autophagy in breast cancer MCF-7cells (35). Bad and BNIP3, two other BH3-only proteins, whenoverexpressed, can induce formation of AV in cancer cells (34,36, 37). Interestingly, Bid, Bad, and BNIP3 are downstream tar-gets of p53, a well characterized tumor suppressor and transac-tivating factor. It has been shown that p53 plays a critical role inthe suppression of tumorigenesis, in part, by the induction ofboth type I and type II PCD (38–41).To identify and characterize novel BH3-only proteins in

AuCD and discover molecular regulators that dictate autoph-agy for death or survival, in this study, we utilized in silico database-mining, functional expression, microscopy strategies, and

lipid binding assay to examine a novel BH3-only protein, apo-lipoprotein L1 (apoL1).

EXPERIMENTAL PROCEDURES

Cell Lines, Chemicals, and Culture Media—DLD-1.TA14 (ap53-null, “Tet-Off” inducible colorectal cancer cell line) andHec50co (a p53-null endometrial cancer cell line) and their cor-responding growthmedium and condition have been describedpreviously (42–44).ATG5�/� andATG7�/�mouse embryonicfibroblast (MEF) cells were gifts from Dr. Mizushima (15) andDr. Komatsu (16), respectively. HEK293 and AD293 cell lineswere purchased fromATCCandClontech, respectively. All celllines except DLD-1 were cultured in Dulbecco’s modifiedEagle’smediumwith 10% fetal bovine serum and 1% antimicro-bial agents as described previously (44).Identification and Sequence Analysis of Novel BH3-only

Proteins by Functional Genomics and BiocomputingApproaches—The known BH3 domain consensus sequenceand a data base-mining strategy were utilized to probe humangenome and proteome data bases for the identification of novelBH3-only proteins as described previously (45). The annotatedhuman open reading frames of candidate genes were down-loaded from the most recent NCBI data base (build 36.2). Inaddition, candidate proteins were assessed for other putativedomains/motifs, utilizing public sequence analysis programsand tools, such as the ExPASy proteomics server and ClustalW,as described previously (45). In addition, protein tertiary struc-ture prediction was obtained from HMMSTR/ROSETTA (46,47), and presentations of tertiary structure were made usingProteinData Bank files createdwithHMMSTR/ROSETTAandPyMOL.Cloning of Human ApoL1 cDNA andGeneration of Inducible

ApoL1 Gene—Human apoL1 was identified as a putative BH3-only protein by in silico data base mining. PCR was used toamplified the 1,194-bp full-length apoL1 cDNA. Subsequently,the sequence-confirmed apoL1 and apoL1 with a C-terminalFLAG tag (apoL1.FLAG) were cloned into pBI-EGFP vector(Clontech). The primers used for amplifying apoL1 were as fol-lows: reverse, 5�-CCGCTCCAGTCACAGTTCTTGGTC-3�(bp �1,197 to �1,183), and forward, 5�-CGACGCGTATGG-AGGGAGCTGCTTTGC-3� (bp �1 to �19). The reverseprimer used for amplifying apoL1.FLAG was 5�-CTAGCTAG-CTCACTTATCGTCGTCATCCTTGTAATCCAGTTCTT-GGTC-3� (bp �1,197 to �1,183). The resulting plasmids wereused to transfect a previously described “Tet-Off” inducible sys-tem in DLD-1.TA14 (44, 45). Stably transfected DLD-1 cellsharboring pBI-apoL1 or pBI-apoL1.FLAG, namely DLD-1.apoL1 andDLD-1.apoL1.FLAG, respectively, were selected innoninductionmedium (D.20) with 20 ng/ml doxycycline (Dox)as described previously (44, 45). We assayed inducible expres-sion of apoL1 transcripts by semi-quantitative RT-PCR andimmunoblot analyses.Site-specific Deletion Mutagenesis—To generate the BH3

domain deletion allele of apoL1, that is to delete the 27 bp thatencode the 9-amino acid BH3 domain (codons 158–166; basepairs �474 to �498), a PCR-based site-directed mutagenesiskit (ExSite, Stratagene) and the deletion-specific primers wereused. Primer sequences were as follows: forward, 5�-AAGGTCC-

ApoL1, a Novel BH3-only Pro-death Protein, Induces Autophagy

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ACAAAGGCACCACC, and reverse, 5�-(p)CCTTCTTATGTT-ATCCTACAGC. The deletion construct pBI-apoL1.dBH3 wasconfirmed by sequencing and was used to construct pAD-apoL1.dBH3 plasmid as described above (44).ApoL1 Adenovirus (AD-ApoL1)—Construction, amplifica-

tion, and assaying for titersof adenoviruses (AD)harboringapoL1and apoL1.dBH3 alleles, and viral infection of mammalian cellswere performed as described previously (44). A variety of cellswere infected with control AD, AD-apoL1, and AD-apoL1.dBH3with a range of concentrations from0.01 to 5multiplicity of infec-tion (or plaque-forming units/cell, whichwas estimated to be 0.2–100 virus particles/cell) for 3 days. Cell numbers, cell death, andsurvival were measured over time by counting the attached cells,and by microscopy analysis in cell morphology (roundup) andincrease of intracellular vacuole formation.Semi-quantitative RT-PCR—Total RNA was isolated from

cells using a Purescript kit (Gentra Systems). RTwas conductedusing random hexamers, and PCR was conducted using gene-specific primers and a standard protocol as described (36, 37).The primers used for PCR were as follows: reverse, 5�-CCGC-TCCAGTCACAGTTCTTGGTC-3� (bp �1,197 to �1,183),and forward, 5�-CAGCAGTACCATGGACTACGG3� (bp�672 to �692).Generation of Anti-peptide Antibodies against Human

ApoL1—To raise anti-peptide antisera specifically againsthuman apoL1, a peptide corresponding to the codons 384–398of apoL1 (ILNNNYKILQADQEL) was used to immunize rab-bits (Anaspec). Antibody was affinity-purified by chromatogra-phy on peptide (antigen)-linked Sepharose and designated asanti-apoL1.Immunoblot Analysis—Total cellular extracts were isolated

and used for immunoblot analysis from indicated cells asdescribed previously (42, 43). Proteins were separated using10% SDS-PAGE (20 �g of protein/lane) and transferred tonitrocellulose membranes, which were then incubated withanti-apoL1, anti-activated caspase-9 (Cell Signaling Technol-ogy), anti-caspase 8 (Cell Signaling), anti-caspase 3, anti-poly-(ADP-ribosyl) polymerase (PARP) (Santa Cruz Biotechnology),anti-LC3 (MBL), and anti-�-actin (Oncogene Research Prod-ucts). Subsequently, blots were incubated with goat anti-rabbitor anti-mouse horseradish peroxidase-labeled secondary anti-bodies (Bio-Rad).Analysis of Dose-dependent, Time Course-dependent Cell

Death—Taking advantage of the inducibility of the Tet-Off sys-tem, dose-dependent and time course-dependent cell deathstudies were conducted. Briefly, stably transfected DLD-1.apoL1 or DLD-1.apoL1.FLAG cells were grown in D.20. Forinduction studies, adherent cells were rinsed in phosphate-buffered saline, refed with the induction medium containinggradually decreasing concentrations of Dox (5 ng/ml, D.5; 1ng/ml, D.1; 0 ng/ml, D.0), and harvested at the indicated timepoints as described previously (31, 38).Assay of Protein Localization by Immunofluorescence

Microscopy—Cells were grown on glass coverslips coated withcollagen (Roche Applied Science). After induction of apoL1 forvarious times, cells were rinsed and then fixed with 4% (v/v)paraformaldehyde solution at room temperature. Coverslipswere rinsed twice with HBSS and blocked with 3% (w/v) BSA in

TBST for30minat roomtemperature.After incubationovernightat 4 °C in 3% (w/v) BSA containing the anti-LC3 antibody (P0036,MBL), coverslipswere rinsedwithHBSS four times and incubatedin 3% (w/v)BSA inTBSTcontainingTexasRed-labeled secondaryantibody (T2767, Invitrogen) for 1 h at room temperature. Finally,coverslips were rinsed with HBSS, mounted onto slides, and sub-jected to fluorescent microscope analysis.Transmission ElectronMicroscopy—Cells on coverslips were

fixed in 3% formaldehyde, 2% glutaraldehyde in 0.1 M sodiumcacodylate buffer (pH 7.4) for 45 min, washed, and finally fixedfor 1 h in 1% osmium tetroxide, 0.5% potassium ferrocyanide incacodylate buffer. The samples were then washed again, dehy-drated with graded alcohol, and embedded in Epon-Aralditeresin (Canemco). The resin was cured at 65 °C for 2 days, afterwhich the glass coverslips were removed from the resin blocksand the samples were sectioned. Ultrathin sections were col-lected on copper grids and examined in anHitachiH-7500 elec-tron microscope. Images were captured and processed with anAMTXR60B camera and ImageCapture software version 5.42.Effect of 3-Methyladenine (3-MA), Wortmannin, and ATGs

on ApoL1-induced Cell Death—To investigate whether apoL1-induced cell death was through autophagy, two different exper-iments were conducted. First, 3-MA (5mM) andwortmannin (1�g/ml), two inhibitors of PI3Ks and autophagy, were added toDLD-1.apoL1 cells 8 h prior to the induction of apoL1. Celldeath was monitored by light microscopy analysis 24 h afterapoL1 induction. Second, wild type (WT), ATG5�/�, andATG7�/� MEF cells were infected with AD-apoL1. Inductionof cell death, overexpression of apoL1, and activation of LC3-IIwere assays 36 h after viral infection.Induction, Immunoprecipitation, and Elution of the

ApoL1.FLAG Fusion Protein—DLD-1.apoL1.FLAG cells weregrown in the inductionmedium for 24 h, rinsed twice with coldphosphate-buffered saline, pelleted, and lysed in lysis buffer (50mM Tris-Cl, pH 7.4, 150 mMNaCl, 1 mM EDTA, and 1% TritonX-100). Total soluble protein extract was incubated with Anti-FLAG-agarose affinity gel (Sigma, F2426) at 4 °C overnightwithgentle agitation. The agarose beadswere thenwashed two timeswith ice-cold TBS (50 mM Tris-HCl and 150 mM NaCl, pH 7.4)and incubated with 400 �l (150 ng/�l) of 3�FLAG peptide(Sigma, F4799) in TBS at 4 °C overnight for eluting apoL1-FLAG fusion protein.In Vitro Lipid Binding Assay—Similar to an immunoblot

analysis, phosphoinositide (PIP), and membrane lipid strips,nitrocellulose membranes pre-spotted with various indicatedlipid species (P-6001, P-6002, Echelon, Salt LakeCity, UT)wereblocked in 3% fat-free BSA for 60 min at room temperature,probed with the purified apoL1.FLAG protein (�100 ng/ml) in3% fat-free BSA in TBST for 1 h at room temperature, followedby primary anti-apoL1 antibody, secondary goat anti-rabbitantibody conjugated with horseradish peroxidase, and ECLdetection and imaging.

RESULTS

ApoL1 Is a BH3 Domain Containing Protein—We utilized a9-residue consensus sequence of the BH3 domain constructedfrom known human Bcl-2 proteins to probe the human pro-teome data base that identified a BH3 domain embedded in the



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apoL1 (GenBankTM accession number NM_003661). We thenretrieved full-length cDNA and protein sequences of apoL1from the National Center for Biotechnology Information

(NCBI) (ncbi.nih.gov) nucleotideand protein sequence data bases.Previously, using the same method-ology, we reported the cloning andcharacterization of a bona fideBH3-only pro-death gene apolipoproteinL6 (apoL6) and showed that overex-pression of apoL6 induces mito-chondria- and caspase 8-mediatedapoptosis (45). Both apoL1 andapoL6 belong to a newly identifiedapolipoprotein L family that sharesignificant sequence identity withinthe predicted amphipathic �-helix.Besides apoL1 and L6, apoL familyhas four other members, L2–5. Ithas been suggested that these pro-teins play important roles in lipidbinding and transport (48–50).Sequence analysis showed thatapoL1 possesses a putative BH3domain (aa 158–166) with 67%sequence identitywith that of apoL6(Fig. 1A). Over all, apoL1 and apoL6possess 27% sequence identity and42% similarity at the polypeptidelevel. In addition to the putativeBH3 domain, apoL1 also possesses aleucine zipper domain (aa 365–392)(Fig. 1A). Sequence alignment of the20-aa putative BH3 domain with anumber of known BH3-only pro-teins (PUMA, BAD, NOXA, andapoL6) and apoL4, further showedthat the 9-aa BH3 domain of apoL1harbors the two important aminoacids, Leu and Asp, conserved in allknown BH3 domains (2, 4). By con-trast, apoL4 does not possess theconserved Asp residue, which issubstituted by an Asn instead (Fig.1B). Tertiary structure prediction,of the 20-aa putative BH3 domain inthe indicated proteins, showed thatthe 9-aa BH3 domain of apoL1 pos-sessed amphipathic �-helical struc-ture, similar to other BH3-only pro-teins of PUMA, BAD, NOXA, andapoL6 (Fig. 1C). In contrast, apoL4does not possess a canonical �-heli-cal structure in that region (Fig. 1C).Finally, sequence analysis suggeststhat apoL1 does not possess BH1,BH2, or BH4 domains. Takentogether, these results suggest that

apoL1 is a putative BH3-only protein.ApoL1 Induces Cell Death in a Variety of Cell Types—To

investigate whether apoL1 induced cell death, we constructed a

FIGURE 1. Sequence and domain analysis of human apoL1. A, polypeptide sequence alignment of human apoL1and apoL6. Over all, apoL1 and apoL6 shows 27% identity and 42% similarity at the aa level. Putative domains inapoL1 include a BH3 domain (aa 158–166) and a leucine zipper domain (aa 365–392). B, sequence alignment of the20-aa region containing putative BH3 domain in known BH3-only proteins (PUMA, BAD, NOXA, and apoL6) andapoL1 and apoL4. The 9-aa BH3 domain are in boldface and the two amino acids, L and D, which are conserved in allknown BH3 domains, are labeled in red with asterisks. ApoL4 does not possess the conserved Asp residue, instead, itis substituted by an Asn (in blue). C, predicted tertiary structure of the 20-aa region containing putative BH3 domainin the indicated proteins. As expected, the 9-aa BH3 domains of known BH3-only proteins, PUMA, BAD, NOXA, andapoL6, show amphipathic �-helical structures. Importantly, the putative BH3 domain of apoL1 also possesses an�-helical domain. In contrast, apoL4 does not possess a complete, canonical �-helical structure in that region.



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Tet-Off apoL1-inducible system inDLD-1 cells (DLD-1.apoL1)and adenovirus harboring wild type apoL1 (AD-apoL1) or aBH3 domain deletion construct of apoL1 (AD-apoL1.dBH3).We first examinedwhether apoL1 expressionwas inducible in atime-dependent manner in stably transfected DLD-1.apoL1cells. Using semi-quantitative RT-PCR and total RNA isolatedfrom DLD-1.apoL1 cells induced for 0, 6, 12, 24, and 48 h, weshowed that there was time-dependent induction of apoL1 ininduced DLD-1.apoL1 cells at the RNA level (Fig. 2A). Weobserved that expression of apoL1 was increased �4-fold 12 hafter the removal of Dox (Fig. 2A). ApoL1 transcripts were alsodetectable in the DLD-1.TA14 control cells, thereby indicatingthat DLD-1 cells have some endogenous apoL1 expression (Fig.2A). To confirm the induction of apoL1 expression at the pro-tein level, we conducted immunoblot analysis on inducedDLD-1.apoL1 cells using anti-apoL1 antibody. As shown in Fig. 2B,the amount of apoL1 protein was greatly increased (�6-fold)12 h after the induction.We then investigated whether overex-pression of apoL1 could promote cell death. As shown in Fig.2C, induction of apoL1 in DLD-1.apoL1 cells had a negativeeffect on cell proliferation significantly reducing the growthrate at 48 hwhen grown in the absence ofDox (D.0; Fig. 2,C, 4thcolumn, andD). Dying/dead cells showed a change in cell mor-phology, increasing intracellular vacuoles, and detachmentfrom culture plates. In contrast, DLD-1.apoL1 cells grown inD.20 exhibited normal growth (Fig. 2, C, 1st column, and D).Similar results were observed inDLD-1.apoL1.FLAGcells (datanot shown). Because apoL1 expression was Tet-Off-regulatedby the concentration of Dox in the culture medium, a cell

growth study was conducted at var-ious concentrations of Dox. In thepresence of gradually increasingDox concentrations, such as 1 ng/ml(D.1) and 5 ng/ml (D.5), cell deathwas decreased in DLD-1.apoL1cells, implying that the cell deathrate was related to the level of apoL1expression. Furthermore, to investi-gate the importance of the BH3domain of apoL1 in the induction ofcell death, we constructed adenovi-rus harboring WT apoL1 (AD-apoL1) or a BH3 domain deletionmutant of apoL1 (AD-apoL1.dBH3)and used those to infect a variety ofcell types, including HEK293 cells.As shown in Fig. 3A, cells overex-pressing green fluorescence proteinalso overexpressed apoL1 protein. Itis evident that greater than 95% ofcells infected with AD-apoL1 weredead, same as those inducing DLD-1.apoL1 cells, 72 h after infection(Fig. 3A, panels b and e). In contrast,AD only or AD-apoL1.dBH3 failedto induce cell death (Fig. 3A, panelsa and d, and c and f, respectively).Immunoblot analysis showed the

protein products of WT apoL1 and apoL1.dBH3 were made ina time-dependent manner and were stable (Fig. 3B, 3rd and 6thlanes for WT apoL1 and 4th and 7th lanes for apoL1.BH3). Inaddition, endogenous expression of apoL1 was very low inHEK293 cells with or without AD infection (Fig. 3B, 1st, 2nd,and 5th lanes). Identical results were observed for the same setof experiments in four different cell lines, DLD-1, HepG2 (livercancer), MCF-7 (breast cancer), and LNCaP, PC3, and DU145(prostate cancer) (data not shown). Thus, AD-apoL1 is a potentinducer of AuCD in a variety of cells and may be a generalautophagy mediator.ApoL1-induced Cell Death Is Not through Caspase-depend-

ent Apoptosis—Because apoL1 and apoL6 have high sequenceand structure similarity and apoL6 induces caspase-dependentapoptosis, we examined whether apoL1-induced cell death wasmediated through apoptosis. Fig. 4 showed that caspases 9 and3 were not activated, and PARP, a well known substrate ofcaspases, was not cleaved during apoL1-induced cell death inDLD-1.apoL1 cells (Fig. 4A). By contrast, apoptotic DLD-1.apoL6 cells showed activation of caspase 9 8 h after apoL6induction, which is consistent with previous findings (45).ApoL1 Induces Activation and Translocation of LC3-II—To

investigate whether apoL1-induced cell death acted throughautophagy, we analyzed apoL1-induced activation and translo-cation of LC3-II to AV, a critical step in autophagic pathway.The amount of LC3-II has been shown previously to correlatewith the number of AV (13, 14, 51). Immunoblot analysis ofLC3 indicated a time-dependent accumulation of LC3-II andincrease in the ratios of LC3-II to LC3-I in induced and auto-

FIGURE 2. Time- and dose-dependent induction of apoL1 and cell death in DLD-1.ApoL1 cells. Inductionof apoL1 expression is time-dependent as indicated by semi-quantitative RT-PCR using total RNA (A) or immu-noblot analysis using total soluble proteins isolated from cells grown in induction medium (D.0) for the indi-cated time (B). C, apoL1 induces dose-dependent cell death. DLD-1.apoL1 cells were cultured in mediacontaining Dox (D; ng/ml) as indicated. Attached cells were harvested and counted after 48 h. D, apoL1induces time-dependent cell death. DLD-1.apoL1 cells were cultured in D.20 (Dox, 20 ng/ml) medium(squares) or D.0 (Dox, 0 ng/ml) medium (triangles), harvested, and counted at the times indicated. Eachexperiment was repeated at least three times. *, p � 0.05, indicates significant difference compared withcontrol by Student’s t test.



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phagic DLD-1.apoL1 cells (Fig. 4B). After induction of apoL1,the ratios of LC3-II and LC3-I were 1:10, 2:1, and 20:1 0, 12, and24 h, respectively. In addition, immunofluorescence micros-copy analysis showed a pattern of punctate staining of LC3-II ininduced DLD-1.apoL1 cells (Fig. 4C, panel b), indicating thetranslocation of LC3-II from cytosol to AV. In contrast, controlDLD-1.apoL1 cells showed a cytosolic staining pattern of LC3(Fig. 4C, panel a), which indicates physiological localization ofLC3-I in normal cells.ApoL1 Induces Increased Formation of AV and Autophagy

Which Can Be Inhibited by 3-MA and Wortmannin—Trans-mission electronmicroscopy (TEM) is one of themost sensitiveand definitive methods to detect autophagic compartments inmammalian cells (52, 53). To further confirm that apoL1-in-duced cell death was indeed through autophagy, we conductedboth light microscopy and TEM analyses. Normal DLD-1.apoL1 cells under light microscopy and TEMwere displayed inFig. 5, A, panel a, and B, panel a, respectively. Induced DLD-1.apoL1 cells with increased expression of apoL1 showed sig-nificant formation of AV under the TEM (Fig. 5B, panels b andc). The magnified images showed an enlarged empty vacuole(Fig. 5B, panel d), a double-membraned AV (Fig. 5B, panel e),and an autophagosome engulfing an electron-dense body (Fig.5B, panel f) in an induced DLD-1.apoL1 cell (Fig. 5B, panel c).Induced DLD-1.apoL1 cells were deformed and slightly

FIGURE 3. BH3 domain deletion construct of apoL1 failed to induce celldeath in DLD-1 cells. A, adenoviruses harboring WT apoL1 (AD-apoL1) andBH3 domain deletion mutant of apoL1 (AD-apoL1.dBH3) were constructedand used to infect DLD-1 cells. Cells overexpressing green fluorescence pro-tein also overexpressed apoL1 protein. AD-apoL1 induced cell death 72 hafter infection (panels b (bright field) and e). In contrast, AD only orAD-apoL1.dBH3 failed to induce cell death (panels a (bright field) and d, and c(bright field) and f, respectively). B, time-dependent protein production of WTapoL1 and apoL1.dBH3. Hours after infection were as indicated. Note thatendogenous expression of apoL1 was very low, almost undetectable, inDLD-1 cells (1st, 2nd, and 5th lanes). Both proteins were stably produced intime-dependent manner (3rd and 6th and 4th and 7th lanes for WT apoL1 andapoL1.dBH3, respectively). �-Actin was used as a loading control.

FIGURE4. ApoL1-induced cell death is through autophagy, not by apoptosis.A, caspases 9 and 3 were not activated, and PARP was not cleaved during apoL1-induced cell death. By contrast, apoL6 induces cell death through apoptosis byactivation of caspases 9 as described previously (45). B, apoL1 induced activationand translocation of LC3. Immunoblot analysis of LC3 in autophagic DLD-1.apoL1cells. Total soluble proteins were isolated at indicated time points from DLD-1.apoL1 cells cultured in induction medium. Times after induction of apoL1 are asindicated. Accumulation of LC3-II in induced DLD-1.apoL1 cells can be seen 12 hafter induction. The ratios between LC3-II and LC3-I were 0.1, 2, and 20, 0, 12, and24 h after induction, respectively. C, immunofluorescence staining of LC3 (red) inDLD-1.apoL1 cells. Panel a, DLD-1.apoL1 cells cultured in normal medium, andpanel b, DLD-1.apoL1 cells cultured in induction medium for 24 h. At least 300cells were analyzed for each experiment.

FIGURE 5. ApoL1 induces accumulation of AV and autophagic cell death,which can be inhibited by 3-MA and wortmannin. A, effect of 3-MA (5 mM)and wortmannin (1 �g/ml) on apoL1-induced cell death was observed bylight microscopy analysis. 3-MA and wortmannin were added to noninduc-tion medium 8 h prior to the induction of apoL1. Panel a, DLD-1.apoL1 cells innoninduction (regular) medium; panel b, in induction medium; panel c, ininduction medium � 3-MA; and panel d, in induction medium � wortmannin.Obviously, 3-MA and wortmannin partially blunt apoL1-induced cell deathin DLD-1.apoL1 cells by 55 and 40%, respectively. All the images were takenby the same inverted microscope (Olympus, CK40) with a MacroFire digitalcamera (OPTRONIC, model S99831). B, electron microscopy analysis of auto-phagic DLD-1.apoL1 cells. Panel a, control DLD-1.apoL1 cells; panels b and c,autophagic DLD-1.apoL1 cells under two different magnifications; panels d–f,magnified views of vacuoles in the cell shown in panel c. Panel d, an enlargedempty vacuole; panel e, a double-membraned structure presumably an auto-phagosome; panel f, an AV, presumably an autolysosome, engulfing an elec-tron-dense body. Scale bars are as indicated. Data are representative of fourexperiments. Each experiment was repeated at least three times.



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enlargedunder the lightmicroscopy (Fig. 5A,panel b). In addition,we examined the effect of 3-MA and wortmannin, two inhibitorsof PI3Ks and autophagy (13, 14, 54), on apoL1-induced cell death.Fig. 5A, panels c and d, showed that 3-MA and wortmannin par-tially blunt apoL1-induced cell death in DLD-1.apoL1 cells cul-tured in induction medium. These data suggest that apoL1-in-duced cell death can be blocked by inhibitors of autophagy.ApoL1 Fails to Induce Cell Death in Autophagy-deficient

MEFs—To further confirm apoL1 induces AuCD, two wellstudied, autophagy-deficient MEFs, ATG5�/� and ATG7�/�,were used. It has been well documented that ATG5 and ATG7are essential for the initiation of autophagosome formation.ATG5 is an acceptor molecule for the ubiquitin-like moleculeATG12, and the proper conjugation of ATG5 with ATG12 isrequired for the elongation of the autophagic membrane (15).ATG7, an E1-like enzyme, together with ATG10, an E2-likeenzyme, catalyze the covalently conjugated complex of ATG5-ATG12. In addition,ATG7 is required for the lipidation/activa-tion of LC3-II and subsequently translocation of LC3-II to the

autophagicmembrane (10, 16). Thus, cells lacking eitherATG5orATG7 are defective in LC3-II activation and autophagosomeformation. MEF cells were infected with AD-apoL1 for 36 h atwhich point induction of apoL1, cell death, and LC3 activationwere assayed. Fig. 6A showed that greater than 90% ofWTMEFcells infected with AD-apoL1 had died, whereas less than 20%of theATG5�/� cells were dead. Fig. 6B showed overexpressionof apoL1 protein was achieved at 36 h after infection of AD-apoL1 in ATG5�/�, ATG7�/�, and WT MEFs. Importantly,apoL1 again induced activation of LC3 II and increased theratio of LC3-II to LC3-I inWTMEF cells (Fig. 6B, 6th lane). Bycontrast, apoL1 failed to induce activation of LC3 and autoph-agy, as expected, in both the ATG5�/� and ATG7�/� cells.These results further indicate that apoL1-induced cell death isautophagy-dependent.Expression of ApoL1 Is Up-regulated by P53 in P53-induced

Cell Death—Previous studies showed that p53, one of the mas-ter regulators in PCD, induces both apoptosis and autophagy(39, 40). To investigate whether the expression of apoL1 wasregulated by p53, we took advantage of two of our previouslyused cancer cell models, DLD-1.p53 and Hec50co, in p53-in-duced apoptosis. Overexpression of p53 was achieved in DLD-1.p53 cells by Tet-Off induction and in Hec50co cells by AD-p53 infection as described previously (44). Immunoblotanalysis showed time-dependent expression of apoL1under theinfluence of p53 in both cell lines. As shown in Fig. 7, inductionof apoL1 was detected 8 and 12 h after p53 overexpression inDLD-1.p53 cells and Hec50co cells, respectively, and 16 and12 h prior to p53-induced cell death in DLD-1.p53 cells andHec50co cells, respectively. This indicates that apoL1 is induc-ible by p53 during p53-induced cell death.ApoL1 Binds Strongly with PA and CL and Less with Some

Phosphoinositides—To explore the binding of apoL1 to lipids,apoL1.FLAG fusion protein was first immunoprecipitated byanti-FLAGantibody and subsequently eluted by 3� FLAGpep-tide. The purified apoL1.FLAG protein was confirmed byimmunoblot analysis using anti-FLAG or anti-apoL1 antibod-ies (Fig. 8A) andwas subsequently used for protein lipid overlayassay. Two membranes pre-spotted with a variety of lipid spe-cies were probed with the apoL1.FLAG fusion protein. Asexpected, Fig. 8B showed that the apoL1.FLAG, but not 3�FLAG itself (negative control), bound to a variety of lipid spe-cies immobilized on the nitrocellulosemembranes in vitro. TheapoL1.FLAG showed strong binding affinity for PA and CL but

FIGURE 6. ApoL1 fails to induce cell death in autophagy-deficientATG5�/� and ATG7�/� MEFs. Wild type (WT), ATG5�/�, and ATG7�/� MEFcells were grown in Dulbecco’s modified Eagle’s medium to 30% confluence,infected with control AD or AD-apoL1 viruses for 36 h, and followed by lightmicroscopy analysis (A). Greater than 90% of WT MEF cells infected with AD-apoL1 had died, whereas less than 20% of the ATG5�/� or ATG7�/� (notshown) cells were dead. B, immunoblot analysis showed overexpression ofapoL1 protein was achieved at 36 h after infection of AD-apoL1 in ATG7�/�,WT, and ATG5�/� MEFs. Importantly, apoL1 again induced activation of LC3 IIand increased the ratio of LC3-II to LC3-I (6th lane). By contrast, apoL1 failed toinduce activation of LC3 in the ATG5�/� and ATG7�/� cells. Of note, it hasbeen reported that MEFs have high endogenous LC3-II (15, 16).

FIGURE 7. Expression of apoL1 is up-regulated by p53 in p53-induced celldeath. Immunoblot analysis of time-dependent expression of apoL1 underthe influence of p53 was assayed. Overexpression of p53 was achieved inDLD-1.p53 cells and in Hec50co cells as described previously (45). Hours afterp53 overexpression are as indicated. Induction of apoL1 was detected 8 and12 h after p53 overexpression in DLD-1.p53 cells and Hec50co cells, respec-tively. �-Actin was used as loading control.



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less affinity with different species of PIPs and other phospho-lipids in the following order: phosphatidylinositol 3,5-bisphos-phate (PI-3,5-P2) � PI4P � PI5P � PI3P � PI-4,5-P2 �PI-3,4,5-P3 � PI-3,4-P2 � 3-sulfogalactosylceramide � phos-phoserine�phosphoglycerol (PG)�phosphoinositol (PI) (Fig.8B). Interestingly, apoL1 did not bind cholesterol, phosphoeth-anolamine (PE), phosphocholine (PC), sphingomyelin (SM),triglyceride (TG), diacylglycerol, sphingosine 1-phosphate,lysophosphatidic acid, and lysophosphocholine.

DISCUSSION

The role of the Bcl-2 family in regulating apoptosis is wellstudied; however, its role in mediating AuCD is still elusive. Inan effort to identify and characterize novel BH3-only proteinsin AuCD, we identified a novel BH3-only pro-death proteinapolipoprotein L1 (apoL1) in silico. Subsequently we showedthatWT apoL1 induces AuCD in a variety of cells, whereas theBH3 deletion construct of apoL1 failed to induce cell death.Interestingly, apoL1 protein has been found both inside the

cells and circulating in the blood. When it is in the serum,apoL1 binds apolipoprotein A-I and is one of the componentsof high density lipoprotein (HDL) particles, suggestingapoL1 has a role in lipid metabolism (48, 49). In addition,protein secondary structure analysis identified four lipid-

binding amphipathic �-helices inapoL1, which may function as acatalyst for intra- and extracellularlipid transfer (48–50). Impor-tantly, we previously reported thatapoL6, a closely related protein ofapoL1 and a BH3-only pro-deathprotein, induces mitochondria- andcaspase-mediated apoptosis (45). Bycontrast we showed apoL4, anothermember of the apoL family not pos-sessing BH3 domain, failed toinduce cell death. Sequence analysisshowed that six of the nine residuesof the BH3 domain are identicalbetween apoL1 and apoL6 (Fig. 1A).In addition, the BH3 domain ofapoL1 showed an amphipathic�-helical structure similar to otherknownBH3domains, including thatof apoL6. Taken together, ourresults indicated that both apoL1and apoL6 are bona fide BH3-onlypro-death proteins. ApoL1 andapoL6 possess conserved primarysequence and tertiary structure;nevertheless, they function differ-ently and nonredundantly in theinduction of PCD. ApoL6 inducesapoptosis (type I), whereas apoL1induces autophagy (type II). It is notunreasonable to postulate that theirinteracting partners, protein and/orlipid species, play important role in

mediating different death-signaling pathways. As a matter offact, we showed that apoL1 has very strong affinity for PA andCL. CL is a dimer of PG and PA and includes four acyl chains,two phosphate groups, and three glycerols. As apoL1 bindsstrongly with PA, it is logical to speculate that apoL1 binds CLthrough its PAmoiety. Interestingly, Fang et al. (55) previouslyshowed that PA is a critical component of mammalian target ofrapamycin (mTOR) signaling. They showed mitogenic stimu-lation of mammalian cells led to a phospholipase D-dependentaccumulation of cellular PA, which was required for activationofmTORdownstreameffectors. PAdirectly interactedwith thedomain in mTOR that is targeted by rapamycin, and this inter-action was positively correlated with the ability of mTOR toactivate downstream effectors. It has also been shown thatmTOR is a negative regulator of autophagy (40, 56). Thus, bind-ing of PA by apoL1 might inhibit mTOR activation and subse-quently induce autophagy. In addition, accumulating evidencesuggests that CL has active roles in mitochondria-mediatedapoptosis (57). Binding of CL with apoL1 may prevent apopto-sis in favor of induction of AuCD.PIPs play critical roles in regulating cellular signaling path-

ways. The homeostatic equilibrium of PIPs determines cellularproliferation, differentiation, trafficking, and PCD (58, 59). Ithas been shown that PI4P is the most abundant member of

FIGURE 8. Interacting lipid species of apoL1.FLAG protein. Affinity-purified apoL1.FLAG fusion protein wasused to conduct lipid overlay assay (see “Experimental Procedures” for details). 3� FLAG peptide solution wasused as a negative control. A, purified apoL1.FLAG was confirmed by immunoblot analysis using both anti-apoL1 and anti-FLAG antibodies. B, apoL1.FLAG bound strongly with CL and PA, followed by PI-3,5-P2, PI4P,PI5P, PI3P, PI-4,5-P2, PI-3,4,5-P3, PI-3,4-P2, 3-sulfogalactosylceramide (3-SGCer), phosphoserine, PG, and PI (veryweak). ApoL1.FLAG did not bind cholesterol (CHO), TG, PE, PC, SM, diacylglycerol (DAG), sphingosine 1-phos-phate, lysophosphatidic acid (LPA), and lysophosphocholine (LPC). Lipid abbreviations are as follows: TG (trig-lyceride), PI (phosphatidylinositol), DAG (diacylglycerol), PI4P (PtdIns(4)P), PA (phosphatidic acid), PI4,5P2(PtdIns(4,5)P2), PS (phosphatidylserine), PI3,4,5P3 (PtdIns(3,4,5)P3), PE (phosphatidylethanolamine), CHO (cho-lesterol), PC (phosphatidylcholine), SM (sphingomyelin), PG (phosphatidylglycerol), 3-SGCer (3-sulfogalactosyl-ceramide (Sulfatide)), CL (cardiolipin), S1P (sphingosine 1-phosphate), LPA (lysophosphatidic acid), LPC (lyso-phosphocholine), PI3,4P3 (PtdIns(3,4)P2), PI3,5P2 (PtdIns(3,5)P2), PI3P (PtdIns(3)P), PI4P (PtdIns(4)P), PI5P(PtdIns(5)P), and Solvent blank (Blank).



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PIPs. Furthermore, PI-3,4,5-P3 and PI-3,4-P2 can induce cellgrowth through the Akt pathway, but PI3P has been shown toinduce autophagy and endosomal trafficking. Our resultsshowed that binding of apoL1 to various species of PIPs mayalter the homeostasis of PIPs leading to the AuCD. Interest-ingly, we found that apoL1 did not bind cholesterol, TG, PC, PE,or SM, themajor lipid components inHDL (Fig. 8B). It has beenshown that apoL1, a minor protein component, can directlyinteract with apoAI, themajor protein component inHDL (48).It is logical to postulate that apoL1,which does not interactwithHDL lipid species, binds and modulates the activities of apoAI.To our best knowledge, apoL1 is the first BH3-only, lipid-bind-ing apolipoprotein that induces AuCD.Interestingly, the genes encoding apoLs are localized to

chromosome 22q12.3-13.1, a high susceptibility locus forschizophrenia. Importantly, expression of apoL1 has beenshown to be significantly up-regulated (�1.41-fold) in thebrains of schizophrenics (60). It will be interesting to seewhether overexpression of apoL1 induces AuCD in centralnervous system cells, and if so what role of apoL1 plays in theetiology of schizophrenia.ApoL1 was recently found to be the trypanosome lytic factor

in human serum. The parasiteTrypanosoma brucei rhodesiensecauses human sleeping sickness in Africa. The molecular basisof this disease is the product of resistance-associated protein,an inhibitory protein that binds specifically to apoL1 to preventcell lysis. Unsequestered apoL1 promotes forming anion chan-nels on lysosomal membranes of trypanosome and subse-quently kills the parasite (61, 62). Thus, in addition to the afore-mentioned functions, apoL1 can also play an important role ininnate immunity.It is well documented that LC3 is required for complete auto-

phagosome formation during autophagic pathway in mamma-lian cells. The combination of alteration of subcellular localiza-tion of LC3-II with increased ratio of LC3-II to LC3-I is one ofthe hallmarks of autophagy (13, 14). This study demonstratesthat apoL1 is a potent and specific inducer of AuCD thatstimulates time-dependent accumulation and translocationof LC3-II, formation of the AV (confirmed by TEM analysis),and subsequently cell death in apoL1-overexpressing cells.Importantly, no apoptotic hallmarks, such as release ofapoptogenic factors from mitochondria or activation ofcaspases, were detected in apoL1-induced cell death. Wealso showed that expression of apoL1 is inducible by p53, awell known tumor suppressor and transactivating factor fre-quently mutated in cancers, which regulates both types ofPCD (38–41). Regarding the role of p53 in autophagy, it hasbeen shown that direct activation of p53 by nutrient starva-tion and serum deprivation induces autophagy in cancercells (40). In addition, damage-regulated autophagy modu-lator, a p53 target gene encoding a lysosomal protein, hasbeen shown to be involved in the induction of autophagy(63). Furthermore, Bad and Bid, two BH3-only proteins anddirect downstream effectors of p53, not only play importantroles in apoptosis but also in autophagy (34, 36, 37). Ourresults suggest that apoL1 is a novel BH3 only protein and isa p53 downstream effector functioning as a molecular deter-minant that dictates autophagy for death (Fig. 9).

Regarding other BH3-only proteins in PCD, Beclin1 interactswith anti-apoptotic Bcl-2 by virtue of its BH3 domain, anamphipathic �-helix, that binds to the hydrophobic cleft ofBcl-2. Interestingly, the association of Beclin1 with Bcl-2 is oflow affinity (1–2 �M) as compared with other BH3-only pro-teins that bind to Bcl-2 (in low nanomolar range) (32–34). Infact, it has been shown that Bad, as well as BH3-mimetic com-pounds such as ABT737, competitively disrupt the inhibitoryinteraction between Beclin1 and Bcl-2 (34). Thus, it is logical tospeculate that when the other BH3-only proteins, such asapoL1 and apoL6, are induced by various stimuli/stresses, theycan displace Beclin1, rendering the cell susceptible to autoph-agy as well as apoptosis, respectively. It will be of interest toinvestigate whether apoL1 and apoL6 interact with anti-deathBcl-2 family members and the functional consequences of theinteractions in PCD. Finally, apoL1 provides an excellentmodelto further study the molecular basis of gene-induced AuCD.

Acknowledgments—We thank Dr. Steven Jett and Tamara Howardfor their excellent EM service at the EM Core Facility, University ofNew Mexico School of Medicine.

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FIGURE 9. Hypothetical model of apoL1-induced autophagic death incancer cells. Expression of apoL1 is inducible by p53, a tumor suppressorgene. Intracellular accumulation of apoL1 alters lipid homeostasis and signal-ing, induces activation and translocation of LC3, and generation of autoph-agic vacuoles leading to cell death. Inhibitors of autophagy, such as 3-MA orwortmannin, blunt apoL1-induced autophagic cell death.



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ApoL1, a BH3-only lipid-binding protein, induces autophagic celldeath

Siqin Zhaorigetu#, Guanghua Wan#, Ramesh Kaini, Zeyu Jiang, and Chien-an A. Hu*Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine,School of Medicine, Albuquerque, NM 87131, USA

AbstractWe recently reported the identification and characterization of a novel BH3-only pro-death protein,apolipoprotein L1 (ApoL1), that, when overexpressed, induces autophagic cell death (ACD) in avariety of cells, including those originated from normal and cancerous tissues. ApoL1 failed to induceACD in autophagy-deficient Atg5−/− and Atg7−/− MEF cells, suggesting that ApoL1-induced celldeath is indeed autophagy-dependent. In addition, a BH3 domain deletion allele of ApoL1 was unableto induce ACD, demonstrating that ApoL1 is a bona fide BH3-only pro-death protein. To furtherinvestigate regulation of ApoL1 expression, we showed that ApoL1 is inducible by interferon-γ andtumor necrosis factor-α in human umbilical vein endothelial cells, suggesting that ApoL1 may playa role in cytokine-induced inflammatory response. Moreover, we observed that ApoL1 is a lipid-binding protein with high affinity for phosphatidic acid and cardiolipin and less affinity for variousphosphoinositides. Functional genomics analysis identified 5 nonsynonymous single nucleotidepolymorphisms (NSNPs) in the coding exons of the human ApoL1 structural gene– all the 5 NSNPsmay cause deleterious alteration of ApoL1 activity. Finally, we discuss the link between ApoL1 andvarious human diseases.

Keywordstrypanosomal lytic factor; apolipoprotein L1 (ApoL1); autophagy; autophagic cell death; BH3-onlyprotein; cardiolipin; lipid binding; PIPs; phosphatidic acid; schizophrenia; SNPs

Macroautophagy (hereafter referred to as autophagy) is a lysosome-dependent mechanism ofintracellular degradation that is used for the turnover and recycling of cytoplasmic constituents.It involves the sequestration of a fraction of cytosol and organelles within a de novosynthesized, double-membraned organelle called the autophagosome, which then fuses withthe endosome and/or lysosome to create the amphisome and autolysosome, respectively.Eventually, the inner membrane of the autophagosome, together with the enclosed cargo, isdegraded by the lysosomal hydrolases.1 Autophagy in mammals serves multiple purposes: itfunctions as a survival mechanism during periods of starvation and stress, it is directly involvedin eliminating aberrant protein aggregates, removing damaged/aged organelles, and defendingagainst pathogens, and it serves an essential role in early neonatal development.2 Evidently,autophagy also functions as type II programmed cell death (PCD), or autophagic cell death(ACD), in multi-cellular organisms.3,4 It has been reported that autophagy is involved in

*Correspondence to: Chien-an A. Hu, Department of Biochemistry and Molecular Biology, MSC08 4670, 1 University of New Mexico,Albuquerque, NM 87131−0001, Tel: (505) 272−8816, Fax: (505) 272−6587; E-mail: [email protected].#These authors contributed equally to the work.Addendum to: Wan G, Zhaorigetu S, Liu Z, Kaini R, Jiang Z, Hu CA. Apolipoprotein L1, a novel Bcl-2 homology domain 3-only lipid-binding protein, induces autophagic cell death. J Biol Chem. 2008; 283:21540−49.

NIH Public AccessAuthor ManuscriptAutophagy. Author manuscript; available in PMC 2009 March 22.

Published in final edited form as:Autophagy. 2008 November 16; 4(8): 1079–1082.

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physiological cell death during development of Drosophila and Dictyostelium discoideum,however, ACD has not yet been observed/demonstrated in mammals in vivo.5,6

In the past two decades, yeast has been used as an experimental model in dissecting themolecular bases of autophagy. Currently 31 ATG (autophagy-related) genes have beenidentified in yeast.3,6 Recent identification and characterization of human orthologs/homologsof yeast ATG genes and the link between autophagy and human diseases further emphasize theimportance of autophagy in health and disease. Deficiency of autophagy correlates withdiseases such as cancer, cardiomyopathies, inflammatory bowel diseases andneurodegenerative diseases.5,7 In addition, autophagy involves unique membrane dynamicsand biosynthesis, requiring de novo synthesis of double-membrane autophagosomes, in whichboth protein and lipid components are required. Regarding protein components, aside fromAtg proteins, Vps34, a class III phosphatidylinositol-3-phosphate kinase (PI3K), the proteinsthat are part of its complex, and the Bcl-2 family members play critical roles in regulatingautophagosome formation. Interestingly, Beclin 1, the mammalian ortholog of yeast Atg6 thatis required for autophagy, is a BH3-only protein. In addition, other Bcl-2 family members,such as Bcl-2, Bax, Bid and BNIP3, regulate autophagy.6,8 By contrast, the lipid compositionof autophagosomal membranes remains unknown because it is technically difficult to isolatepure autophagosomes. It is evident however that some lipid species, for example,phosphatitylinositol-3-phosphate or PI3P, are required for autophagosomal membranes.1,9

In an effort to identify and characterize novel BH3-only proteins in ACD, and discovermolecular regulators that dictate autophagy for death or survival, we examined a novel BH3-only protein, apolipoprotein L1 (ApoL1). We show that wild-type ApoL1 induces ACD in avariety of cells, whereas a BH3 deletion allele of ApoL1 fails to induce cell death, indicatingthat ApoL1 is a bona fide BH3-only pro-death protein. We further demonstrate that ApoL1 isa potent and specific inducer of ACD that stimulates time-dependent accumulation andtranslocation of lipidated LC3-II, formation of autophagosomes and subsequently cell death.We also show that ApoL1 is inducible by p53 during p53-induced cell death. p53, a well-knowntumor suppressor, regulates both apoptosis and ACD.4 Interestingly, a recent study shows thatp53 plays a dual role in the control of autophagy: on the one hand, nuclear p53 can induceautophagy through transcriptional effects, whereas on the other hand, cytoplasmic p53 mayact as a master repressor of autophagy.10 Our results suggest that ApoL1 is a novel BH3-onlyprotein and is a p53 downstream effector functioning as a molecular determinant thatdetermines whether autophagy plays a role in cell death.4 Importantly, ApoL1 has been foundonly in humans and African green monkeys (NCBI Entrez Gene, ApoL1), suggesting thatApoL1-induced ACD is primate-specific.

Employing a protein-lipid overlay assay, we demonstrate that ApoL1 is an intracellular lipid-binding protein that binds strongly with phosphatidic acid (PA) and cardiolipin (CL), and withless affinity with different species of phosphoinositides (PIPs) in the following order PI(3,5)P2>PI4P>PI5P>PI3P>PI(4,5)P2>PI(3,4,5)P3>PI(3,4)P2.4 PIPs are phosphorylated derivativesof the membrane phospholipids phosphatidylinositols and play critical roles in regulatingreceptor signaling, cytoskeleton function and membrane trafficking. They are highlyconcentrated and localized in distinct pools in the plasma membrane, endosomes, nucleus andautophagosomal membranes, where they function as ligands, adaptors or docking sites for PIP-binding proteins. In addition, the homeostatic equilibrium of PIPs determines cellularproliferation, differentiation and PCD.11,12 Vps34 is required for autophagy, implying anessential role of its product PI3P in this process.13 Recently, Obara and colleagues9 showedthat PI3P is highly enriched and delivered to autophagosomal membranes but not as a cargoenclosed in autophagosomes, implying direct involvement of PI3P in autophagosomeformation. They also report a possible enrichment of PI3P on the inner autophagosomalmembranes compared to the outer membrane.9 Regarding binding with PIPs, we show that

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ApoL1 possesses higher affinity for PI(3,5)P2, PI4P, PI5P, and PI3P, implying that intracellularaccumulation of ApoL1 may alter the homeostasis of PIPs leading to ACD. In addition, weconducted a protein domain homology search and found no homology between ApoL1 andany known PIP-binding domains, including FYVE (Fab1, YOTB, Vac1 and EEA1), PX (Phoxhomology), PH (Pleckstrin homology), ENTH (Epsin N-terminal homology), FERM(Band4.1, Ezrin, Radixin, and Moesin), GRAM (Glucosyltransferase, Rab-like GTPaseActivator, and Myotubularins), ANTH (AP180/CALM), and WD-repeats of WIPIs, humanorthologs of yeast Atg18 and/or Atg20.7,14,15 In fact, no specific binding domain for PI(3,5)P2 has been identified thus far. Taken together, ApoL1 may represent a novel class of PIP-binding protein. Importantly, in autophagy, at least 2 Atg proteins are involved in PIP synthesis(Atg6 and Atg14) and 5 Atg proteins bind PIP (Atg18, Atg20, Atg21, Atg24 and Atg27).1,7Evidently, yeast mutant strains deficient in PIP binding will be useful for functionalcomplementation and biochemical analysis of human ApoL1.

Interestingly, ApoL1 also binds PA and CL. PA is a mitogenic messenger that serves as acritical component of mTOR signaling. PA directly interacts with the domain in mTOR thatis targeted by rapamycin, and this interaction positively correlates with mTOR's ability toactivate downstream effectors. mTOR is a negative regulator of autophagy;16,17 thus, bindingof PA by ApoL1 might inhibit mTOR activation and subsequently induce autophagy. Inaddition, CL, a negatively charged phospholipid abundant in the mitochondrial innermembrane, is important in maintaining protein (for example cytochrome c) and membraneintegrity of mitochondria. During apoptosis, CL functions as the docking site for t-Bid and Badand induces mitochondrial membrane permeability and cytochrome c release.18,19 In addition,CL relocates to the plasma membrane and other subcellular organelles during cell death.20Sequestering of PA and CL with ApoL1 may alter the homeostasis between survival and deathleading to ACD. To our knowledge, this is the first BH3-only protein with lipid binding activitythat, when overproduced intracellularly, induces ACD.

Roles of ApoL1 in human diseases

African Sleeping sicknessThe parasites Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense causehuman sleeping sickness in Africa. It has been known for over a century that human serumcontains trypanosomal lytic factor (TLF) that can lyse most of the invading trypanosomespecies. TLF, residing in a minor subclass of human high-density lipoprotein particles, containsapolipoprotein A-I (ApoA-I) and two primate-specific proteins, ApoL1 and hepatoglobin-related protein (Hrp), both of which are toxic to trypanosomes.21,22 The molecular basis ofthis disease is that pathological trypanosomes produce the serum resistance-associated protein(SRA), an inhibitory protein that binds specifically to ApoL1 to prevent trypanosomal lysis.Unsequestered and engulfed ApoL1 promotes the formation of anion (Cl−) channels onlysosomal membranes of trypanosomes and induces osmotic swelling of lysosomes due tomassive influx of chloride ions from the cytoplasmic compartment, and subsequently kills theparasite.21 In addition to ApoL1 and Hrp, Wildener and colleagues23 recently provided invitro evidence that hemoglobin is a cofactor of human TLF that increases the affinity of TLFfor its receptor, and therefore the internalization efficiency of TLF to the parasite. Moreimportantly, ApoL1 alone is sufficient to confer trypanolytic activity in transgenic mice butboth ApoL1 and Hrp are required to increase the specific activity of TLF.24 It is worth notingthat a recent case report documents that an Indian patient whose serum lacked trypanolyticactivity due to the absence of ApoL1 was infected by a serum-sensitive T. evansi, a strain thatwould normally not infect humans. Mutation analysis shows that the patient possesses twoframeshift mutations in both ApoL1 alleles, further emphasizing the critical role of ApoL1 ininnate immunity against trypanosomal infection.22 Nevertheless, it is evident that ApoL1-

Zhaorigetu et al. Page 3

Autophagy. Author manuscript; available in PMC 2009 March 22.

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